Proteome analysis of motor proteins in rat parotid acinar cells

采用蛋白质组分析和Western blotting技术对大鼠腮腺中的马达蛋白进行分析，研究马达蛋白和细胞骨架在分泌颗粒和分泌囊泡分泌淀粉酶过程中的作用，RT-PCR结果表明，大鼠腮腺中存在KifC 1、C2、C3、C4和5 B（泛在驱动蛋白）。在腮腺分泌颗粒膜上检测到肌球蛋白、泛在驱动蛋白（Kif 5 B）、动力蛋白/动力蛋白p150/动力蛋白p50复合物。微管蛋白和肌动蛋白也被检测到的分泌颗粒膜。肌动蛋白与β-肾上腺素能刺激的分泌有关，而微管蛋白与β-肾上腺素能刺激的分泌无关。肌球蛋白不参与淀粉酶的分泌过程。提示腮腺腺泡细胞的β-肾上腺素能过程可能涉及微管和运动蛋白非依赖性机制。另一方面，微管和肌球蛋白与毒蕈碱刺激的淀粉酶释放有关。这些结果报道在Arch Oral Biol（49：975-82，2004）中。通过粗颗粒/囊泡级分的Redi格拉德梯度离心分离分泌囊泡级分。微管蛋白和肌动蛋白的分泌囊泡部分检测。用抗KIFC 2抗体在50 KDa处观察到免疫反应性条带，但不在适当位置。动力蛋白/动力肌动蛋白复合物中未检测到的馏分。在非刺激条件下的淀粉酶释放可能归因于分泌囊泡。非刺激性淀粉酶的释放被茉莉花素减少，但不被秋水仙素减少。肌球蛋白ATP酶抑制剂2，3-丁二烯一肟2，3-丁二烯一肟不影响非刺激的淀粉酶分泌。细胞骨架和马达蛋白在非刺激性分泌和调节性分泌中发挥不同的作用。

Motor proteins in rat parotid glands were analyzed by proteome analysis and Western blotting.The roles of the motor proteins and the cytoskeletons were investigated in the process of amylase secretion from the secretory granules and the secretory vesicles.RT-PCR indicated that KifC1, C2, C3, C4, and 5B (ubiquitous kinesin) were present in the rat parotid glands. Kif 5B was degradated to show the two protein bands about 80KDa and 50KDa in the cytosol fraction prepared from rat parotid gland homogenate.Myosins, ubiquitous kinesin (Kif 5B), dynein/dynactin p150/dynactin p50 complex were detected on the secretory granule membranes. Tubulin and actin were also detected on the secretory granule membranes. Actin was related to the secretion by β-adrenergic stimulation, but tubulin was not. Myosin was not involved to the process of the amylase secretion. These indicated that a microtubule- and motor protein-independent mechanism may be involved in the β-adrenergic process in parotid acinar cells. On the other hand, microtubules and myosin were related to the muscarinic stimulated-amylase release. These results were reported in Arch Oral Biol (49:975-82, 2004).Secretory vesicle fraction was separated by Redi Grad -gradient centrifugation of the crude granule/vesicle fraction. Tubulin and actin were detected in the secretory vesicle fraction. Immunoreactive band was observed with anti-KIFC2 antibody at 50KDa but not at the adequate position. Dynein/dynactin complex was not detected in the fraction. Amylase release under non-stimulated conditions may be attributed to the secretory vesicles. The non-stimulated amylase release was decreased by jasplakinolide but not decreased by colchicine. The myosin ATPase inhibitor 2,3-butanediene monoxime2,3-butanediene monoxime did not affect the non-stimulated amylase secretion. The cytoskeleton and the motor protein played different roles in the non-stimulated and the regulated secretion.